Naturally occurring interferons (IFNs) are species-specific proteins, often glycoproteins, produced by various cells upon induction with viruses double-stranded RNAs, other polynucleotides, antigens and mitogens Interferons exhibit multiple biological activities such as antiviral, antiproliferative, immunomodulatory and anticellular functions. At least three distinct types of human interferons have been identified and characterized in terms of their antiviral, antigrowth and activation of natural killer cell (NK) activities. They are produced by leukocytes, lymphocytes, fibroblasts and the immune system and are classified as .alpha., .beta. and .gamma. interferons. These are reported to be different proteins coded for by distinct structural genes.
Native human .beta.-interferon (HuIFN-.beta.) is generally produced by superinducing human fibroblast cultures with Poly-IC (Polyriboinosinic acid and polyribocytidylic acid) and isolating and purifying the HuIFN-.beta. thus produced by chromatographic and electrophoretic techniques. Proteins or polypeptides which exhibit native .beta.-interferon-like properties may also be produced using recombinant DNA technology by extracting poly-A-rich 12S messenger RNA from virally induced human cells, synthesizing double-stranded cDNA using the mRNA as a template, introducing the cDNA into an appropriate cloning vector, transforming suitable microorganisms with the vector, harvesting the bacteria and extracting the HIFN-.beta. therefrom. Nagola, S., et al., Nature (1980) 287:411; Yelverton. E., et al., Nuc Acid Res (1981) 9:731; Steuli. M., et al., Proc Natl Acad Sci (U.S.A.) (1981) 78:2848: European Patent Applications Nos. 2803, published May 6, 1981: 321134, published July 15, 1981; 34307 published August 26, 1981; and Belgian Patent No. 837397, issued July 1, 1981, describe various currently used methods for the production of .beta.-interferon employing recombinant DNA techniques. The expressed proteins of polypeptides have been purified and tested and have been found to exhibit properties similar to those of native IFNs. Bacterially produced IFNs thus appear to have potential therapeutic use as antiviral and antitumor agents and the production of IFNs by such bacterial fermentations is expected to yield sufficiently large quantities of IFN at a relatively low cost of clinical testing.
Further HuIFN-.beta. genes have been altered by, for example, oligonucleotide-directed mutagenesis to produce IFN-.beta. protein analogs thereof, such as the human recombinant cysteine-depleted or cysteine-replaced interferon-.beta. analogs (muteins) disclosed in U.S. Pat. No. 4,588,585, issued May 13, 1986 to Mark et al. Specifically disclosed in that patent is the recombinant IFN-.beta. wherein the cysteine at position 17 is replaced by the neutral amino acid serine. That IFN-.beta. analog is IFN-.beta..sub.ser17.
Microbially produced rIFN-.beta. to which this invention applies is not glycosylated and is produced in a denatured state. It is insoluble and, when expressed at high levels, it precipitates intracellularly in the form of "refractile" or "inclusion" bodies which appear as bright spots visible within the enclosure of the cell under a phase contrast microscope at magnifications down to 1000 fold.
The heretofore available methods for recovering microbially produced rlFN-.beta. from the organisms that produce it are described below.
Procedures for recovering and purifying bacterially produced IFNs are described in U.S. Pat. Nos. 4,450,103; 4,315,852; 4,343,735: and 4,343,736; and in Derynch et al., Nature (1980) 287:193-197, and Scandella and Kornberg, Biochemistry (1971) 10:4447. With these methods the IFN generally is not produced in a sufficiently pure form and in sufficiently large quantities for clinical and therapeutic purposes, and the resulting IFN preparations produced by recombinant DNA techniques have residual amounts of chemicals, such as sodium dodecyl sulfate (SDS) and other surfactants or precipitants used in the extraction and purification steps.
U.S. Pat. No. 4,620,928 describes a process for recovering rIFN-.beta. from an rIFN-.beta.-producing microorganism in which the cell is disrupted; non-rIFN-.beta. proteins are extracted selectively from the disruptate using an aqueous solution of a chaotropic agent such as urea or guanidine; the rIFN-.beta. is solubilized in a denaturing environment, such as guanidine, containing a reducing agent; the reducing agent, after a suitable time, is removed from the solution; the rIFN-.beta. is subjected to a controlled oxidation; and the oxidized rIFN-.beta. is renatured, optionally followed by a combination of HPLC and gel filtration steps.
Commonly owned U.S. Pat. Nos. 4,530,787 and 4,572,978 describe techniques for carrying out the controlled oxidation step referred to above. The former patent uses o-iodosobenzoic acid as an oxidizing agent and the latter uses Cu.sup.+2 cation as an oxidation promoter.
Copending, commonly owned U.S. applications Ser. Nos. 749,951, filed June 26, 1985 and 843,997, filed Mar. 25, 1986 and entitled "Process for Recovering Refractile Bodies Containing Heterologous Proteins from Microbial Hosts" disclose methods for recovering and purifying refractile bodies of various proteins from E. coli. To isolate the refractile material, the processes initially involve disrupting the cell wall and membrane of the host cell, removing greater than 99% by weight of the salts from the disruptate. redisrupting the desalted disruptate, adding a material to the disruptate to create a density or viscosity gradient in the liquid within the disruptate, and separating the refractile material from the cellular debris by high-speed centrifugation. The refractile protein is then solubilized with a solubilizing agent such as SDS, chromatographed to remove high molecular weight contaminants, oxidized, and purified by HPLC, ultrafiltration, and gel filtration. The process of the present invention uses some of the techniques disclosed in these applications to obtain the solubilized IFN-.beta. starting material.
In addition, U.S. Pat. Nos. 4,511,502: 4,511,503; 4,512,922 and 4,518,526: and EPA No. 114,506 describe a similar procedure for recovering heterologous proteins in general from refractile bodies. In such processes, the oxidation and renaturation of the recombinant protein are carried out in a single step.
Copending. commonly owned U.S. application Ser. No. 923,423, filed Oct. 27, 1986 and entitled "Pharmaceutical Compositions of Recombinant Beta-Interferon and Formulation Processes" discloses the use of certain non-ionic detergents for solubilization of recombinant IFN-.beta.. Many of the detergents and solubilizing agents disclosed therein are useful in the process of the present invention.
Purification procedures for IFN-.beta. produced recombinantly in bacteria have been published by Lin, L.S., et al., Methods in Enzymology (1986) 119:183; and by Moscheva, J. A., et al., ibid., p. 177.